Development of a μ-control strategy for filamentous fungi based on viable biomass measurement

Abstract

Filamentous fungi are used for large scale production of antibiotics, enzymes, organic acids and other industrial relevant compounds. An example is the production of ß- lactam antibiotics by the fungus Penicillium chrysogenum. Prior studies have found from steady state carbon limited chemostat cultivations carried out at different dilution rates, that the specific penicillin production rate (qp) depends on the growth rate (-). The obtained steady-state qp(-) relations are not suitable for a fed-batch with constant feeding, due to a changing growth rate in time. Nevertheless, studies showed a quadratic interaction between - and qp in fed-batch processes and proved it with a fed-batch cultivation. Growth of pellets is mainly characterized by their radius. Pellet morphology is a result of environmental conditions and pellet age. Both, radius and pellet morphology can influence the diffusion coefficient and the penetration depth of oxygen into the pellet. The relationship between morphology and productivity has been observed in several studies, however indifferent statements can found in the literature. The main goals of this work were i. the development of a method to monitor viable biomass ii. the development of a control strategy for cell specific growth rate iii. investigations of changes in the morphology of filamentous fungi Therefore four fermentations were performed by applying different feeding profiles in order to develop a method to monitor viable biomass. After a batch on complex substrate for spore germination and biomass production, the cell broth was used for the inoculation of two subsequently performed fed-batches in two bioreactors. Following, six fermentations were performed investigating a new cultivations strategy targeting the control of the specific growth rate by applying the before developed strategy of viable biomass monitoring. For all performed cultivations morphological analysis of the most important parameters were conducted. Through the application of different feeding profiles different amount of viable biomass was produced. Therewith, a method to estimate viable biomass based on capacitance measurements in dual frequency mode (100 kHz - 580 kHz, background frequencies 15650 kHz and 19490 kHz) could be established. In a next step, the established method was applied online / at-line. Moreover, the cultivation strategy was further adapted over several performed cultivations. First, low biomass concentrations were targeted by means of a dilution step on the beginning of the C-limited fed-batch. Due to decreased product titer levels, the cultivation strategy needed to be further adapted. For following cultivations the fed-batch started immediately with a C-limitation in order to reach low biomass concentrations on the beginning of the fed-batch. Finally, through the start of the fed-batch with a C-limitation low biomass concentrations and increased product titer could be achieved. Hence, this adapted cultivation strategy was used for all following cultivations. The development of a control strategy for cell specific growth, could be achieved based on the improved at-line measurement and cultivation strategy of the before developed method. Moreover, due to further investigations of changes in the morphology of filamentous fungi, correlations between the amount of pellets and the specific growth rate, as well as the morphological parameter fullness (tightness of the core) and roughness (amount of hyphae on the outside) could be found.